Summary

Pitx2, a paired-related homeobox gene that encodes
multiple isoforms, is the gene mutated in the haploinsufficient Rieger
Syndrome type 1 that includes dental, ocular and abdominal wall anomalies as
cardinal features. Previous analysis of the craniofacial phenotype of
Pitx2-null mice revealed that Pitx2 was both a positive
regulator of Fgf8 and a repressor of Bmp4-signaling,
suggesting that Pitx2 may function as a coordinator of craniofacial
signaling pathways. We show that Pitx2 isoforms have interchangeable
functions in branchial arches and that Pitx2 target pathways respond
to small changes in total Pitx2 dose. Analysis of Pitx2
allelic combinations that encode varying levels of Pitx2 showed that
repression of Bmp signaling requires high Pitx2 while maintenance of
Fgf8 signaling requires only low Pitx2. Fate-mapping studies
with a Pitx2 cre recombinase knock in allele revealed that
Pitx2 daughter cells are migratory and move aberrantly in the
craniofacial region of Pitx2 mutant embryos. Our data reveal that
Pitx2 function depends on total Pitx2 dose and rule out the
possibility that the differential sensitivity of target pathways was a
consequence of isoform target specificity. Moreover, our results uncover a new
function of Pitx2 in regulation of cell motility in craniofacial
development.

Introduction

Pitx2 is a paired-related homeobox gene that was shown to
be the gene mutated in Rieger Syndrome type I (RGS I)
(Semina et al., 1996), an
autosomal dominant, haploinsufficient disorder that includes tooth
abnormalities as one of its primary features
(Flomen et al., 1998). The
craniofacial defects in individuals with RGS I, that have one half dose of
Pitx2, include dental hypoplasia, anodontia vera, abnormally shaped
teeth and a flattened midface (Amendt et
al., 2000). Individuals with RGS I also have ocular anterior
chamber disorders, which often result in glaucoma and umbilical abnormalities
(Semina et al., 1996).
Pitx2 plays a central role in left right asymmetry
(Capdevila et al., 2000;
Harvey, 1998) and is a
component of Wnt-β-catenin signaling in pituitary and cardiac outflow
tract development (Kioussi et al.,
2002). Experimental evidence supports the idea that the dominant
genetics of RGS I results from haploinsufficiency; however, there is evidence
for a dominant negative mechanism in a subset of patients
(Saadi et al., 2003;
Saadi et al., 2001).

Investigation of Pitx2 function using loss-of-function approaches
in mice has shown that Pitx2 plays an important role in early stages
of tooth development (Gage et al.,
1999; Kitamura et al.,
1999; Lin et al.,
1999; Lu et al.,
1999). Pitx2-null mutant embryos had arrested tooth
development at placode or bud stage. Consistent with a haploinsufficient
mechanism, tooth phenotypes were observed in Pitx2 null
+/– mice (Gage et al.,
1999). Early epithelial-mesenchymal signaling was intact in
Pitx2-null embryos as suggested by the presence of a condensed dental
mesenchyme (Lin et al., 1999;
Lu et al., 1999). Expression
of markers such as Shh and mesenchymal Bmp4 and
Msx1 also supported the idea that tooth initiation and specification
occurred but tooth germ expansion failed in Pitx2-null embryos
(Lin et al., 1999;
Lu et al., 1999). In situ also
showed that Bmp4 expression was expanded, while Fgf8 failed
to be expressed or was downregulated in oral epithelium of Pitx2-null
embryos (Lin et al., 1999;
Lu et al., 1999). Taken
together, these data suggest that the initial events in tooth development
occurred in the absence of Pitx2, subsequent signaling events were
deranged resulting in a premature extinction of Fgf8 expression and
failure of demarcation of Bmp4 expression to dental epithelium. These
experiments uncovered an early function for Pitx2 in tooth
morphogenesis but failed to address any later role for Pitx2 in
craniofacial development.

The Pitx2 gene encodes three isoforms, Pitx2a, Pitx2b and
Pitx2c in mice and a fourth Pitx2 isoform, Pitx2d,
has been identified in humans (Cox et al.,
2002). The different isoforms are generated by both alternative
splicing and alternative promoter usage
(Shiratori et al., 2001)
(Fig. 1A,B) and have both
overlapping and distinct expression patterns. All Pitx2 isoforms have
a common C terminus and distinct N termini
(Fig. 1A). Pitx2c is
the asymmetrically expressed isoform while Pitx2a, Pitx2b and
Pitx2c isoforms are co-expressed in head mesoderm, oral ectoderm,
eye, body wall and central nervous system
(Kitamura et al., 1999;
Liu et al., 2001;
Schweickert et al., 2000;
Smidt et al., 2000).
Pitx2c, but not Pitx2a or Pitx2b, is expressed in
hematopoietic stem cells (Degar et al.,
2001). Co-expression of Pitx2 isoforms is found in the
three developmental fields that are most frequently affected in individuals
with RGS I: eyes, teeth and anterior abdominal wall.

The observation that Pitx2 regulated two fundamentally important
signaling pathways in craniofacial morphogenesis raised the possibility that
haploinsufficiency observed in humans and mice was a consequence of
differential sensitivity of these important target pathways to total
Pitx2 dose. An alternative idea, suggested by multiple Pitx2
isoforms with overlapping expression in developing teeth, was that
Pitx2 function in craniofacial development was a consequence of
distinct isoform function. For example, it is conceivable that one
Pitx2 isoform functions to repress Bmp4 while a separate
isoform maintains Fgf8 expression. In addition, Pitx2
isoforms have been shown to form heterodimers in vitro suggesting that
Pitx2 isoform heterodimers may have distinct target genes
(Cox et al., 2002).
Overexpression of a Pitx2 engrailed repressor
(enr) fusion protein in left lateral plate of chick
embryos revealed that Pitx2c enr but not a Pitx2a
enr fusion could interfere with endogenous Pitx2c
function (Yu et al., 2001),
consistent with the idea that Pitx2 isoforms have distinct target
genes. Experiments performed in Xenopus and zebrafish, as well as
tissue culture studies, support the idea that Pitx2 isoforms have
distinct targets (Cox et al.,
2002; Essner et al.,
2000; Faucourt et al.,
2001; Suh et al.,
2002).

We investigated Pitx2 isoform function in craniofacial
morphogenesis by analyzing craniofacial phenotypes of isoform-specific
deletions. We used Pitx2 alleles that encode differing levels of
Pitx2 to investigate the requirements for total Pitx2 dose
in craniofacial morphogenesis (Liu et al.,
2001). Our results show that Pitx2 isoforms have
interchangeable function in craniofacial development and that signaling
pathways that are regulated by Pitx2 respond differently to changes
in total Pitx2 dose. The Fgf8 maintenance pathway uses low
Pitx2 doses, while Bmp4 repression requires high
Pitx2 doses. Our findings uncovered downstream functions for
Pitx2 in tooth development and fate mapping experiments with a
Pitx2 cre recombinase knock-in allele revealed that Pitx2
daughter cells are migratory. Movement of Pitx2 daughters was
aberrant in Pitx2 mutants, suggesting that Pitx2 regulates
cell movement in craniofacial primordia.

lacZ staining and histology

Mouse embryos were fixed in Bouin's, dehydrated and embedded in paraffin
wax. Sections were cut (7-10 μm) and stained with Hematoylin and Eosin.
lacZ staining was as previously described
(Lu et al., 1999).

Results

Pitx2 isoforms are co-expressed in oral and dental
epithelium

The Pitx2 δabcnull allele, a homeobox
deletion, removes function of all isoforms, while theδ
abhypoc and δab alleles delete the
Pitx2a and Pitx2b specific exons and leave Pitx2c
intact (Fig. 1A,C). Theδ
abhypoc allele, which retains PGKneomycin, encodes
less Pitx2c function than the δab allele in which
PGKneomycin was removed (Liu et al.,
2001). We generated a deletion of the Pitx2c isoform
(Liu et al., 2002), theδ
c allele, that was a replacement of the
Pitx2c-specific exon 4 with a LoxP flanked PGKneomycin. In the finalδ
c allele, PGKneomycin has been removed by crossing to the
CMVcre deletor strain (Liu et
al., 2002) (Fig.
1C). To study the developmental progression of Pitx2
daughter cells (see below), we generated Pitx2δ
abccreneo, a Pitx2 cre recombinase knockin
allele (Fig. 1B; see Materials
and methods). We introduced cre into Pitx2 exon 5 that
resulted in a Pitx2 null allele and expressed cre in the
same spatiotemporal pattern as endogenous Pitx2 (see below). Excision
of the PGKneomycin cassette by crossing to the rosa26 eFlp deletor strain
resulted in the Pitx2 δabccre allele.

We studied Pitx2a and Pitx2b isoform expression using theδ
abhypoc and δab alleles that contain
a lacZ knock-in into Pitx2 exon 2 and deletes Pitx2
exon 3 (Fig. 1C-F). As
lacZ was introduced into exon 2, this analysis provides information
about Pitx2a and Pitx2b specific expression but does not
distinguish between these two isoforms because Pitx2a uses exon 2 and
Pitx2b uses both exon 2 and exon3
(Fig. 1A). We used RT-PCR to
distinguish between Pitx2a and Pitx2b expression (see
below). We also performed in situ analysis using a Pitx2c probe. At
10.5 dpc, lacZ was expressed uniformly throughout the oral ectoderm,
while at 14.5 dpc, lacZ expression was found in dental epithelium and
primary enamel knot of cap stage tooth
(Fig. 1D-F). Using a
Pitx2c probe for in situ, we detected Pitx2c expression
throughout the 10.5 dpc oral ectoderm (Fig.
1G). At 14.5 dpc, Pitx2c was expressed in dental
epithelium similarly to Pitx2a and Pitx2b
(Fig. 1H,I). To distinguish
between Pitx2a and Pitx2b isoform expression in oral
ectoderm, we performed RT-PCR with a primer set that distinguished between
Pitx2a, Pitx2b and Pitx2c. We identified all three isoforms
in the mandibular arch epithelium at 10.5 and 12.5 dpc
(Fig. 1J). These data suggest
that the Pitx2a, Pitx2b and the Pitx2c isoforms are
coexpressed in oral ectoderm and, at later stages, within tooth epithelial
structures.

Pitx2 isoforms have interchangeable functions in tooth
development

Co-expression of Pitx2 isoforms suggests a number of possibilities
for the regulation of target pathways by Pitx2. It is possible that
Pitx2 isoforms would regulate distinct target genes in tooth
formation or Pitx2 isoforms may have redundant functions. Isoform
co-expression also supports the idea that some Pitx2 target genes
have a requirement for Pitx2 heterodimers
(Cox et al., 2002). To address
these ideas, we analyzed forming teeth ofδ
ab–/– andδ
c–/– embryos.

As a control, we analyzed teeth of δab;δc
mutant embryos. We reasoned that this allelic combination should encode near
normal levels of all Pitx2 isoforms, albeit from different
chromosomes, and should be functionally similar toδ
abcnull heterozygous embryos. Analysis of coronal
and sagittal sections through the teeth of δab;δ
c embryos at 14.5 and16.5 dpc revealed that tooth development
was normal (Fig. 2A-D). From
this, we conclude that the δab and δc alleles
encode adequate levels of Pitx2 isoforms to support normal tooth
development.

To test the idea that Pitx2 isoforms had distinct target genes and
thus distinct functions in tooth development, we analyzed the teeth ofδ
ab–/– embryos at two timepoints, 16.5
dpc and 18.5 dpc. We found that teeth of δab homozygous mutant
embryos that lack Pitx2a and Pitx2b are normal suggesting
that there is redundant function between the Pitx2a, Pitx2b and
Pitx2c isoforms in tooth development or that Pitx2c has the
major role in tooth development (Liu et
al., 2001) and Fig.
2G,H,J,K). Sections through Pitx2c mutant teeth at 16.5
and 18.5 dpc revealed normal molar tooth morphology suggesting that Pitx2
a, Pitx2b and Pitx2c isoforms have redundant function in tooth
morphogenesis (Fig. 2G,I,J,L).
These data argue against an absolute requirement for either Pitx2
isoform-specific target genes or Pitx2 isoform heterodimers in
branchial arch morphogenesis and tooth development. These results suggest that
common Pitx2 target genes are differentially regulated by total
Pitx2 dose (Table
1).

Previous data suggested that Fgf8 expression was absent in
Pitx2 δabcnull homozygous mutants
(Lu et al., 1999) but was
diminished only in embryos homozygous mutant for an independently generated
Pitx2-null allele (Lin et al.,
1999). One idea to explain this discrepancy is that Fgf8
expression was induced but not maintained in Pitx2-null mutant
embryos. To determine if Pitx2 was required for the maintenance of
Fgf8 expression, we examined Fgf8 expression in
Pitx2-null mutants at earlier timepoints than previously reported. In
9.5 dpc δabcnull homozygous mutant embryos, low
levels of Fgf8 mRNA was expressed in the oral ectoderm
(Fig. 3A,B). Sectioning
revealed that the Fgf8 expression domain was restricted to a small
region of oral ectoderm at the proximal aspect of the mandibular process in
Pitx2 δabcnull homozygous mutants when
compared with wild-type embryos (Fig.
3C,D). In the absence of Pitx2, the majority of the oral
ectoderm loses the competency to express Fgf8, suggesting that
Pitx2 has a role in the demarcation of the Fgf8 expression
domain to the proximal aspect of the mandibular and maxillary processes. At
later timepoints, Fgf8 expression is lost in Pitx2-null
mutants (Lu et al., 1999) (see
below).

We examined expression of genes that are proposed Fgf8 targets in
mandibular mesenchyme. Lhx6 expression was shown to be dependent on
Fgf8 function as Lhx6 failed to be induced in mutants with
an oral ectoderm specific inactivation of Fgf8
(Trumpp et al., 1999). In
Pitx2 δabcnull mutants, Lhx6
expression was reduced (Fig.
3E,F). The residual Lhx6 expression in the Pitx2δ
abcnull embryos was in the proximal mandible near
the region where Fgf8 was expressed in the Pitx2δ
abcnull mutant embryos
(Fig. 3D). Expression of
Pitx1, normally expressed in the oral ectoderm and proximal
mandibular mesenchyme, has been shown to be induced by implantation of an
Fgf8 bead (St Amand et al.,
2000). Pitx1 expression was reduced in the proximal
aspect of the Pitx2 δabcnull mutant
mandibular arch mesenchyme at 10.5 dpc
(Fig. 3G,H). Expression of
Dlx2 in mandibular mesenchyme has also been shown to be upregulated
by Fgf8 bead implantation (Thomas
et al., 2000). We found that the mesenchymal expression of
Dlx2 was reduced in Pitx2 δabcnull
mutants (Fig. 3I,J). As
previous data suggested that induction of Pitx1 and Dlx2
expression was independent of Fgf8, our results suggest that
Fgf8 functions to maintain pitx1 and dlx2
expression in the mandibular mesenchyme
(Trumpp et al., 1999).
Expression of endothelin 1 (Edn1), also dependent on Fgf8
function, was downregulated in the mandibular arch ectoderm of Pitx2δ
abcnull mutants
(Fig. 3K,L). It is notable that
expression of Lhx6, Pitx1, and Dlx2 in the maxillary
primordial of Pitx2 δabcnull mutants was
also reduced; however, further experiments are necessary to rule out the
possibility that this was secondary to reduction in the outgrowth of the
forming maxilla (Fig.
3E-J).

We noted that Dlx2 was still expressed in the caudal aspect of the
Pitx2 mutant mandibular mesenchyme
(Fig. 3I,J). As Pitx2
expression is restricted to the rostral mandibular arch ectoderm, continued
expression of Dlx2 in caudal mandibular mesenchyme suggested that
Fgf8 signaling from the caudal aspect of the mandibular ectoderm was
intact in the Pitx2 δabcnull mutant embryos
and that patterning of the mandibular process was disrupted in the
Pitx2 δabcnull mutants. Goosecoid
(Gsc), an Fgf8 responsive homeobox gene, is normally
expressed in the caudal mandibular arch mesenchyme. Caudal Gsc
expression is normally maintained via a Fgf8 repressive pathway that
inhibits Gsc expression in the rostral mandibular process
(Tucker et al., 1999). We
reasoned that if maintenance of Fgf8 signaling was disrupted in
Pitx2 δabcnull mutants, then Gsc
expression should be expanded rostrally. We found that Gsc expression
was weakly expanded in a subset of Pitx2δ
abcnull mutants embryos
(Fig. 3M,N), while in the
remainder of mutant embryos Gsc expression was caudally restricted
(data not shown). The incomplete penetrance of expanded Gsc
expression suggests that in the subpopulation of Pitx2 mutant embryos
with correct Gsc expression, the early Fgf8 expression was
sufficient to specify the correct Gsc expression domain.

Correct patterning of the mandibular mesenchyme is necessary for formation
of Meckel's cartilage (Tucker et al.,
1999). Based on the weak expansion of Gsc expression, we
expected that Pitx2-null mutants would have a weak Meckel's cartilage
phenotype. To assess this, we performed whole-mount cartilage staining on
Pitx2 δabcnull mutants and control
wild-type littermate embryos. The Pitx2δ
abcnull mutants had a variable deficiency of
Meckel's cartilage supporting the notion that rostral caudal polarity of the
mandibular process was weakly affected by loss of Pitx2 function
(Fig. 3O,P). Taken together,
these data suggest that in the absence of Pitx2, Fgf8 expression in
oral ectoderm fails to be maintained. In the absence adequate Fgf8
signaling, Fgf8-dependent signaling to underlying mesenchyme is
reduced leading to defective mandibular arch rostral caudal polarity.

Differential sensitivity of Pitx2 target pathways to changes
in total Pitx2 dose

We investigated whether repression of Bmp signaling by Pitx2 was
also rescued in theδ
abcnull;δabhypoc allelic
combination that encodes low levels of Pitx2 function. To assess
expansion of Bmp signaling, we examined Bmp4 expression in oral
ectoderm of 10.5 dpc Pitx2 mutant embryos. In contrast to the
Fgf8 signaling pathway, Bmp repression required high levels of
Pitx2 function. In Pitx2δ
abcnull–/– embryos Bmp4
expression was expanded laterally in mandibular process ectoderm
(Fig. 4A,B)
(Lu et al., 1999). In
wild-type embryos, Bmp4 expression is found in the medial mandibular
process and the distal aspect of the ectoderm of the maxillary process at 10.5
dpc (Fig. 4B,E). In
Pitx2δ
abcnull;δabhypoc andδ
abcnull;δab allelic combinations,
Bmp4 expression in the mandibular process was weakly expanded.
Moreover, in the maxillary process ectoderm of Pitx2δ
abcnull;δabhypoc andδ
abcnull;δab mutants, Bmp4
expression failed to be distally restricted and was detected all the way to
the junction with the mandibular process
(Fig. 4C-G).

We examined expression of Msx1 and Msx2 that are
mesenchymal targets of Bmp signaling
(Barlow and Francis-West, 1997;
Vainio et al., 1993). In
Pitx2 δabcnull–/– embryos andδ
abcnull;δabhypoc andδ
abcnull;δab allelic combinations,
expression of Msx2 (Fig.
4H-K) and Msx1 (Fig.
4L-O) was expanded proximally in the mandibular and maxillary
processes. These data also revealed that expression of Msx1 and
Msx2 was more obviously expanded than the Bmp4 ligand,
particularly in the mandibular process in Pitx2 mutant allelic
combinations. We noted that expression of Msx1 and Msx2 was
expanded in the branchial arch mesenchyme that probably contributes to the
developing heart in some Pitx2 mutant embryos
(Fig. 4K,O). Taken together,
these results suggest that maintenance of Fgf8 expression and
repression of Bmp-signaling pathways have distinct requirements for
total Pitx2 dose in the branchial arches (summarized in
Table 1).

Pitx2 regulates tooth orientation and cap formation

We investigated the tooth morphology of theδ
abcnull;δabhypoc andδ
abcnull;δab allelic combinations
using histological analysis. Sections through 18.5 dpc wild-type, andδ
abcnull;δab mutant embryos revealed
well-formed molars. We found that in the δabcnull;δ
ab embryos, the orientation of the molar tooth was abnormal
(Fig. 5A,C,E,G). Inδ
abcnull;δabhypoc 18.5 dpc
mutant embryos, analysis of serial sections revealed that molar teeth were
absent (Fig. 5B,F). As
lacZ marks cells fated to express Pitx2a and
Pitx2b, serving as a marker of dental epithelium, we performed
lacZ staining on serial cryosections from heads of 14.5 dpc
Pitx2 allelic combinations. In δab+/–
and δabcnull; δab embryos,
well-formed cap stage molar teeth were clearly evident with lacZ
staining (Fig. 5I,J). Inδ
abcnull; δabhypoc mutant
embryos, the dental lamina invaginated but failed to form the dental cap
(Fig. 5K). In Pitx2δ
abcnull homozygous mutant embryos, tooth
development arrested at the placode or bud stage. The molar phenotype inδ
abcnull; δabhypoc
embryos, with a more developed dental lamina, suggests that tooth development
progressed further than in δabcnull mutant embryos.
These data show that as the dose of Pitx2 decreases there is evidence
of increasingly severe defects in tooth morphogenesis.

From these results, we conclude that Pitx2 has a late function in
molar orientation and in morphogenesis of the cap stage tooth. The
intermediate tooth phenotypes observed in theδ
abcnull; δabhypoc andδ
abcnull; δab mutants most probably
reflects a direct role for Pitx2 in morphogenesis of dental
epithelium. Although it is possible that expression of Fgf8 in the
Pitx2 δabcnull;δ
abhypoc and δabcnull;δ
ab oral ectoderm is inadequate to completely rescue molar
tooth development, the expression of Pax9 and Barx1 in
dental mesenchyme of these allelic combinations suggests that Fgf8
signaling to mesenchyme is intact in these mutant embryos and argues that
Pitx2 directly regulates epithelial morphogenesis.

Our previous data revealed that Pitx2 functioned to regulate local
cell movement in heart development (Liu et
al., 2002). To determine if a similar mechanism was at work in
craniofacial development, we used the δabccre knock
in allele and the Gtrosa 26 reporter mouse to follow the movement of
Pitx2 daughter cells within the first branchial arch. At 9.5-11.0
dpc, cre expression was detected in the oral ectoderm in bothδ
abccre+/– andδ
abccre; δabcnull embryos,
although by 11.0 dpc cre expression was diminished in theδ
abccre; δabcnull embryos
(Fig. 6A,B and not shown).
Cre expression was restricted to oral ectoderm and was not found in
facial ectoderm or epithelium lining the oral cavity
(Fig. 6C-E). Fate mapping with
the GtRosa26 reporter showed that Pitx2 daughters were
detected in the oral ectoderm, periocular mesenchyme, guts, heart and body
wall (Fig. 6F,G).

In the craniofacial region, Pitx2 daughters moved outwards from
the oral ectoderm to the facial ectoderm in both wildtype and mutant embryos
(Fig. 6H-K). As cre
mRNA expression was restricted to oral ectoderm, these data reveal that
lacZ-positive migrating cells were Pitx2 daughters that had
extinguished Pitx2 expression. There were differences in the pattern
of daughter migration in Pitx2-null mutant compared with wild-type
embryos. In wild-type embryos, Pitx2 daughters moved a short distance
to cover the outer aspect of the mandibular and maxillary process. Some
Pitx2 daughters also contributed to the nasal process of wild-type
embryos (Fig. 6H,J). In
Pitx2 mutant embryos, daughter cells moved aberrantly in a dorsal
direction just inferior to the eye and failed to contribute to the mutant
nasal process (Fig. 6I,K).

Pitx2 daughters extensively populated the floor and roof inside
the forming mouth (Fig. 6L-O).
In Pitx2 mutants, fewer daughter cells populated the oral cavity roof
as compared with wild type (Fig.
6N-Q). Pitx2 daughters contributed to Rathke's pouch and
dental epithelium, of both the wild type and mutant although in the
Pitx2 mutant tooth morphogenesis was arrested
(Fig. 6N-S and not shown).
These data reveal that Pitx2 daughter cells exit the oral ectoderm
and contribute to both facial ectoderm and the ectoderm lining the oral cavity
and Pitx2 function is necessary for correct deployment and expansion
of daughter cells.

Discussion

In craniofacial development, the mechanisms that organize growth and
morphogenesis of the branchial arches remain poorly understood. We
investigated Pitx2 isoform function in craniofacial morphogenesis
using Pitx2 exon-specific deletions. Analysis of Pitx2
allelic combinations encoding different levels of Pitx2 also
uncovered the influence of variations in total Pitx2 dose on
Fgf8 and Bmp4 signaling
(Table 1). Our data indicate
that Pitx2 isoforms have interchangeable function in craniofacial
development and that Pitx2 target pathways have distinct requirements
for total Pitx2 dose. Reduced Pitx2 levels resulted in
unbalanced interplay between Fgf8 and Bmp4 signaling
pathways in craniofacial morphogenesis. We found that Pitx2 daughter
cells are migratory, eventually populating intraoral and facial ectoderm, and
that Pitx2 function is required for this movement. We provide
evidence that Pitx2 connects overall growth and morphogenesis of the
first branchial arch through a mechanism involving differential sensitivity of
target pathways to total Pitx2 dose.

Deletion of Fgf8 in oral ectoderm revealed a role for
Fgf8 in survival and outgrowth of mandibular mesenchyme
(Trumpp et al., 1999), while
pharmacological suppression of Fgf signaling in explants suggested that Fgf
functioned primarily by signaling to the underlying mesenchyme
(Mandler and Neubuser, 2001).
Bead implantation also suggested an early role for Fgf8 in
establishing the maxillo-mandibular region of the chick embryo
(Shigetani et al., 2000).
Importantly, antagonistic interactions between Fgf and Bmp signaling has been
implicated in proximodistal mandibular arch patterning, placement of tooth
organ formation and determination of the maxillo-mandibular region of the
early embryo (Neubuser et al.,
1997; Shigetani et al.,
2000; Tucker et al.,
1998).

In contrast to Fgf8, high doses of Pitx2 are required for
repression of Bmp signaling. In theδ
abcnull; δabhypoc andδ
abcnull; δab mutants, expression of
Bmp4 was expanded in maxillary ectoderm while Msx1 and
Msx2 expression was expanded in mesenchyme of both maxillary and
mandibular processes. Thus, expression of the Bmp target genes was more
significantly expanded than expression of Bmp4 ligand. This may
reflect the induction of a signal relay cascade in the mandibular process. It
is also interesting to note that Dpp has been shown to act as a
classical morphogen in the wing imaginal disc of Drosophila
(Entchev et al., 2000;
Teleman and Cohen, 2000).

We found that in δabcnull;δ
abhypoc and δabcnull;δ
ab Pitx2 mutants components of Bmp4 and Fgf8
signaling pathways, such as Msx1 and Barx1, are co-expressed
in mandibular mesenchyme. Previous work suggested an antagonistic interaction
between these two signaling pathways
(Neubuser et al., 1997;
Tucker et al., 1998). It is
likely that in the Pitx2 mutant allelic combinations, Bmp signaling
is only weakly expanded and this is insufficient to antagonize expression of
Barx1 in mandibular mesenchyme.

These data provide insight into the normal function of Pitx2 in
regulating gene expression. The Fgf8 pathway and the Bmp
suppression pathway have different requirements for total Pitx2 dose.
As Pitx2, Fgf8 and Bmp4 are co-expressed in many cells of
the oral ectoderm, one can envision a mechanism where Pitx2 would
directly regulate Fgf8 and Bmp4 expression. In this model,
one idea to explain the different requirements for Pitx2 dose in
regulating Bmp4 and Fgf8 would be that the regulatory
regions of Bmp4 and Fgf8 contain different numbers of
high-affinity Pitx2-binding sites, a mechanism suggested to underlie
the haploinsufficiency of individuals with Holt-Oram syndrome that are
heterozygous for tbx5 (Bruneau et
al., 2001). Thus, Pitx2 target genes with more
Pitx2-binding sites would require higher doses of Pitx2 for
correct levels of gene expression. However, this model is complicated by in
vitro observations showing that Pitx2 can cooperatively bind DNA
(Dave et al., 2000;
Wilson et al., 1993),
suggesting that low levels of Pitx2 can form higher order complexes
on DNA. It is likely that there are other mechanisms, such as interaction with
co-factors, to constrain or augment the ability of Pitx2 to activate
target genes. Further experiments are necessary to rule out the possibility
that Pitx2 indirectly regulates the Fgf8 and Bmp4
pathways.

Pitx2 in tooth morphogenesis and cell movement in
craniofacial development

Pitx2-null embryos have arrest of tooth development at the placode
or bud stage (Gage et al.,
1999; Lin et al.,
1999; Lu et al.,
1999). In the Pitx2 δabcnull;δ
abhypoc and δabcnull;δ
ab embryos, molar tooth morphogenesis was partially rescued in
that an invaginated dental lamina formed without a cap or the orientation of
the dental cap was abnormal. Our in situ studies showed that Fgf8 was
expressed in the oral ectoderm of δabcnull;δ
abhypoc and δabcnull;δ
ab embryos. Moreover, expression of Pax9 was also
detected in the prospective dental mesenchyme and Barx1 was expressed
in proximal mandibular mesenchyme of these embryos revealing that Fgf
signaling to mandibular mesenchyme is intact in the Pitx2 hypomorphic
embryos. Although expanded Bmp signaling could account for tooth
defects in the δabcnull;δ
abhypoc and δabcnull;δ
ab embryos, the abnormal tooth morphology was not suppressed
by reducing Bmp4 dose using a Bmp4-null allele (W.L. and
J.F.M., unpublished). Based on these data, we favor the notion that
Pitx2 regulates tooth morphogenesis through a pathway that is
distinct from Fgf8 and Bmp4 signaling, although further
experiments are required to investigate these ideas.

Our fate-mapping studies show that Pitx2 daughter cells move from
oral ectoderm to populate facial and inner oral cavity ectoderm.
Pitx2-expressing cells make a decision to extinguish Pitx2
and become motile. It may be that Pitx2 expression promotes cell
compaction or inhibits cell motility. It is notable that one of the phenotypes
of the Pitx2-null embryos was failure of compaction and
differentiation of the periocular mesenchyme
(Lu et al., 1999).
Fgf8 signaling was implicated in cell movement as Fgf8-null
embryos had defects in cell migration through the primitive streak. Analysis
of Xenopus sprouty2, an inhibitor of Fgf signaling, revealed that Fgf
signaling in Xenopus regulated both mesoderm induction and convergent
extension movements (Nutt et al.,
2001). Thus, it is plausible that Pitx2 regulates cell
movement in the craniofacial primordia through an Fgf8-mediated
pathway.

A direct connection of Pitx2 to cytoskeleton and morphogenetic
movement has been made by the observation that Pitx2 controls Rho
GTPase activity by regulating expression of the guanine nucleotide
exchange factor, Trio (Wei and
Adelstein, 2002). It has recently been proposed that
Pitx2 is a target of canonical Wnt β-catenin signaling pathway
in pituitary and cardiac development
(Kioussi et al., 2002). This
work uncovered a genetic interaction between Pitx2 and dishevelled 2,
a Wnt pathway branchpoint, in the heart. Other studies showed that
Rho family GTPases are downstream components of non-canonical planar
cell polarity (PCP) pathway (Habas et al.,
2003; Strutt et al.,
1997; Winter et al.,
2001). Although further experiments are required, our data showing
that Pitx2 daughters are migratory supports the idea that
Pitx2 may be a component of a non-canonical Wnt pathway in
craniofacial development.

Pitx2 and the phenotypic heterogeneity of Rieger syndrome
I

The phenotypes in individuals with Rieger syndrome with PITX2
mutations are heterogeneous. Our data reveal that slight changes in
Pitx2 dose can have a large influence on resulting phenotypes. This
is illustrated most clearly by comparing theδ
abcnull; δabhypoc andδ
abcnull; δab mutants that have only
slight changes in Pitx2 activity but dramatic differences in tooth
morphogenesis (Liu et al.,
2001). Many organ systems, such as heart and lungs, cannot
distinguish between these small differences in Pitx2 activity
(Liu et al., 2001).

The isoform deletions of Pitx2 reveal functional redundancy
between isoforms in tooth development. These data are consistent with the
observation that all Pitx2 mutations detected in individuals with
Rieger syndrome are in regions common to all isoforms
(Alward, 2000;
Kozlowski and Walter, 2000;
Priston et al., 2001;
Saadi et al., 2001). Our data
suggest that the Pitx2 N terminus does not have a significant
function in tooth morphogenesis because this region is not conserved between
Pitx2a, Pitx2b and Pitx2c. This differs from pituitary and
skeletal muscle where the N terminus has an influence on Pitx2
function (Kioussi et al.,
2002; Suh et al.,
2002). It is also clear that Pitx1 functions
cooperatively with Pitx2 in pituitary organogenesis and limb
development (Marcil et al.,
2003). As Pitx1 is co-expressed with Pitx2 in
developing teeth, it will be interesting to investigate potential cooperative
functions of Pitx1 and Pitx2 in oral and dental
epithelium.